US4970158A - Beta amylase enzyme product, preparation and use thereof - Google Patents

Beta amylase enzyme product, preparation and use thereof Download PDF

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Publication number
US4970158A
US4970158A US07/015,239 US1523987A US4970158A US 4970158 A US4970158 A US 4970158A US 1523987 A US1523987 A US 1523987A US 4970158 A US4970158 A US 4970158A
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enzyme
amylase
optimum
sub
beta amylase
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US07/015,239
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English (en)
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Helle Outtrup
Barrie E. Norman
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Novozymes AS
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Novo Industri AS
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Assigned to NOVO INDUSTRI A/S, A DANISH CORP. reassignment NOVO INDUSTRI A/S, A DANISH CORP. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: NORMAN, BARRIE E., OUTTRUP, HELLE
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Assigned to NOVOZYMES A/S reassignment NOVOZYMES A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NOVO NORDISK A/S
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2425—Beta-amylase (3.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus

Definitions

  • This invention relates to a novel ⁇ -amylase enzyme product, a process for its preparation, compositions containing said ⁇ -amylase in combination with other enzymes, and the use of the ⁇ -amylase or such compositions in the production of high maltose syrups.
  • ⁇ -amylase is the name traditionally given to exacting maltogenic amylases which catalyze the hydrolysis of 1,4- ⁇ -glucosidic linkages in amylose, amylopectin and related glucose polymers Maltose units are successively removed from the non-reducing chain ends in a stepwise manner until the molecule is degraded or, in the case of amylopectin, until a branch point is approached. The maltose released has the ⁇ anomeric configuration hence the name ⁇ -amylase.
  • ⁇ -form of maltose will in aqueous solutions, isomerize spontaneously to a mixture of the ⁇ - and ⁇ -forms. Prolonged reaction of ⁇ -amylase on amylopectin or partially degraded amylopectin, results in the formation of ⁇ -limit dextrin i.e. material which is not susceptible to further hydrolysis by ⁇ -amylase.
  • ⁇ -amylases may be used to produce maltose containing syrups of use in the confectionary, baking, and brewing industries.
  • ⁇ -amylases are conventionally used in combination with debranching enzymes in order to increase the maltose content in the product by hydrolyzing the 1,6- ⁇ -glucosidic bonds in the ⁇ -limit dextrins.
  • ⁇ -amylases have been isolated from various plants and microorganisms (W. M. Fogarty and C. T. Kelly, Progress in Industrial Microbiology, vol. 15, p. 112-115, 1979). These ⁇ -amylases are characterized by having optimum temperatures in the range from 40° C. to 65° C. and optimum pH in the range from 4.5 to 7.
  • Debranching enzymes exhibit for the most a pH optimum in the range from 3.5 to 6 and are thus more active in an acid solution.
  • the pH chosen is usually a compromise between the pH optimum of each of the chosen enzymes or the enzymes are used in succession.
  • the vegetal ⁇ -amylases further have the disadvantage that their production require large amounts of raw material and a large energy consumption for the extraction and processing, whereas microbial ⁇ -amylases can be produced on a large scale at relatively low costs.
  • the present invention is based upon the discovery that a novel microbial extracellular ⁇ -amylase (21-51 ⁇ -amylase) having such properties is produced from a newly isolated Bacillus strain NCIB 11608.
  • FIG. 1 is a plot of relative activity of the maltogenic enzyme against temperature
  • FIG. 2 is a plot of relative activity against pH.
  • the present invention provides a ⁇ -amylase enzyme product which comprises a novel thermostable ⁇ -amylase having the following characteristics:
  • the present invention provides a process for the preparation of the above thermostable ⁇ -amylase enzyme product which process comprises the cultivation of the above mentioned Bacillus , strain NCIB 11608 or variants or mutants thereof productive of this enzyme in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, optionally followed by recovery of the ⁇ -amylase enzyme product
  • the present invention provides a process for the production of high purity maltose syrups wherein starch is treated with the novel ⁇ -amylase enzyme product in an aqueous medium.
  • the industrial process of producing maltose syrups comprises liquefying starch, then saccharification with a maltose producing enzyme, and optionally with an enzyme cleaving the 1,6-branching points in amylopectin, for instance pullulanase or isoamylase.
  • the present invention provides an active enzymatic composition comprising the novel ⁇ -amylase product in combination with at least one debranching enzyme.
  • the present invention provides a process for the production of high purity maltose syrups, wherein starch is treated with the novel active enzymatic composition of the invention.
  • 21-51 ⁇ -amylase hydrolyzes amylopectin, glycogen, and amylose, releasing only ⁇ -maltose.
  • branched polysaccharides such as amylopectin and partially hydrolyzed amylopectin 21-51 ⁇ -amylase forms ⁇ -limit dextrins, which can be hydrolyzed by glucoamylase, isoamylase, pullulanase, or the like.
  • 21-51 maltogenic amylase differs from other Bacillus ⁇ -amylases in the following respects:
  • the microorganism capable of producing the ⁇ -amylase according to the present invention was selected by means of its ability to grow on an agar substrate prepared as follows:
  • Tryptone (10 g), amylopectin (CPC snowflake 10 g), Bacto agar (40 g), and deionized water (1000 ml) are mixed aseptically at 55° C. with an equivalent amount of a salt solution of the following composition:
  • the pH of the salt solution being adjusted to 3.0 with 10N sulphuric acid.
  • the isolated microorganism was deposited with the National Collection of Industrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, on Mar. 15, 1983 and accorded the reference number NCIB 11608 NCIB, being an international depository authorized under the Budapest Treaty of 1977, affords permanence of the deposit and accessability thereto to the public in accordance with rules 9 and 11, respectively, of said treaty.
  • NCIB National Collection of Industrial Bacteria
  • Torry Research Station Aberdeen, Scotland, on Mar. 15, 1983
  • NCIB 11608 NCIB being an international depository authorized under the Budapest Treaty of 1977, affords permanence of the deposit and accessability thereto to the public in accordance with rules 9 and 11, respectively, of said treaty.
  • Rod-shaped bacteria having a diameter of 0.6 to 0.8 ⁇ m.
  • the spores are oval to cylindrical in form, placed centrally to subterminally and do not cause swelling of the sporgania.
  • the Bacillus NCIB 11608 do not grow on conventionally used media with a pH above 6.
  • the optimal conditions of growth for this bacterium are pH 4.8 to 5.8 and a temperature of 30° to 37° C.
  • the maximal temperature for growth of the bacterium is 45° C.
  • BA-1 salts comprise.
  • pH is adjusted to 3.0 with 10N H 2 SO 4 prior to autoclaving.
  • pH in the ready-mixed tryptone basis medium is 4.8 to 5.2.
  • the standard growth medium for the proliferation of NCIB 11608 contains 0.5% amylopectin and no yeast extract. Vegetative cells of NCIB 11608 do often contain bubbles which in a phase contrast microscope appear as refractive spheres, and which are not coloured when the cells are lightly stained by safranin or methylene blue.
  • the morphological and biochemical properties are very much like those that characterize Bacillus megaterium, but the properties of the beta amylase are very far from those that till now have been described for Bacillus megaterium beta amylase
  • the microorganism is believed to belong to the Bacillus acidopullulyticus complex described in U.S. Pat. No. 4,560,651.
  • One ⁇ -amylase unit is defined as the amount of enzyme which under standard conditions (temperature 60° C., pH 4.5, and reaction time 30 minutes) produces reducing sugar corresponding to 1 ⁇ mol maltose per minute
  • 0.5 ml 2% starch in 0.1M acetate buffer is incubated with 100 ⁇ l of the enzyme dissolved in deionized water containing 0.5-2 ⁇ U per ml. The reaction is stopped after 30 minutes by addition of 0.3 ml 0.5N NaOH, and the mixture diluted in a ratio of 1:10 with deionized water.
  • the content of reducing sugar is then determined by means of the Somogyi method (Somogyi: J. Biol. Chem., 153, p. 375-80 (1944)).
  • a Bacillus strain capable of producing the ⁇ -amylase of the present invention is usually propagated on a solid substrate prior to its cultivation under aerobic conditions in a suitable fermentation medium.
  • Both media contain assimilable sources of carbon and nitrogen besides inorganic salts optionally together with growth promoting nutrients, such as yeast extract
  • the fermentation is typically conducted at 30°-37° C. and at a pH of 5-6 and preferably kept approximately constant by automatic means.
  • the enzyme is excreted into the medium.
  • the ensuing fermentation broth may be freed of bacterial cells and debris therefrom together with other solids, for example by filtration.
  • the supernatant containing the enzyme may be further clarified, for example by filtration or centrifugation, and then concentrated as required, for example by ultrafiltration or in an evaporator under reduced pressure to give a concentrate which, if desired, may be taken to dryness, for example by lyophilization or spray-drying.
  • the maltogenic amylase of the present invention can be purified from a continuous fermentation culture broth as follows:
  • the powder is dissolved in 15 mM acetate buffer, pH 5.0 and dialysed against 15 mM acetate buffer pH 5.0 until the conductivity is about 1 mS.
  • the dialyzate is then applied to a cation exchanger CM-sepharose Cl-6B which has been equilibrated with the same buffer.
  • the amylase passes through the column whereas 60% of the remaining proteins is withheld by the ion-exchanger.
  • the pH of the effluent from this column is adjusted to 4.0 with acetic acid and the eluate is subsequently applied to a CM-sepharose Cl-6B column equilibrated with 15 mM acetate buffer pH 4.0. Under these circumstances the amylase is adsorbed by the ion-exchanger. The enzyme is then eluated with acetate buffer of pH 4.0 with increasing ionic strength. The enzyme activity in the eluate follows the protein content in a symmetrical peak.
  • FIG. 1 graphically illustrates the relative activity plotted against temperature (substrate 2% soluble starch, pH 4.5 (0.1M acetate), 30 minutes reaction time), and
  • FIG. 2 graphically illustrates the relative activity plotted against pH (temp. 60° C., substrate 2% soluble starch, 30 minutes reaction time, McIlvaine buffer).
  • 21-51 ⁇ -amylase has an activity optimum at pH 4.5 of about 70° C. and that its pH optimum is in the range of 4.0-5.0. More than 60% of the maximum activity is still found at 80° C.
  • the 21-51 culture was grown at 37° C. for 2 days on the following agar:
  • the pH of the salt solution being adjusted to 3.0 with 10N H 2 SO 4 .
  • the fermentation was carried out in a BIOFLO® (New Brunswick, Canada) fermentor with 1 liter working volume.
  • the fermentation was started up with 100 ml of the above inoculum and the substrate dosage was started after 24 hours at 30° C.
  • the pH was adjusted to 5.5 ⁇ 0.2 with 3% sulphuric acid and the temperature was kept at 30° C. ⁇ 0.2.
  • Substrates for saccharificaton were prepared by redissolving a 7DE (dextrose equivalent) spray-dried maltodextrin in deionized water and making up to approximately 30% DS (dry substance).
  • the saccharification experiments were carried out in standard 500 ml laboratory batch reactors Aliquots of the substrates were heated to 55° C. or 60° C., the pH adjusted to an initial value of 5.5, and 5 ⁇ U or 20 ⁇ U/g DS were then added.
  • Samples were withdrawn after 4, 24, 48, 96, and 168 hours and heated in boiling water for 10 minutes to inactivate the enzyme After cooling the samples were filtered and treated with a mixedbed ion exchange resin (Bio-Rad® AG 501 ⁇ 8(D)) to remove ash and soluble N before being analyzed by HPLC and gel chromatography.
  • a mixedbed ion exchange resin Bio-Rad® AG 501 ⁇ 8(D)
  • DP refers to the degree of polymerization, DP1 being monosaccharide(s) e.g., glucose, DP2 being disaccharides, e.g., maltose, DP3 being traisaccharides and DP4 being a saccharide oligomer with a degree of polymerization of four or more

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
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  • Biomedical Technology (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
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US07/015,239 1986-02-19 1987-02-17 Beta amylase enzyme product, preparation and use thereof Expired - Fee Related US4970158A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK077086A DK160563C (da) 1986-02-19 1986-02-19 Beta-amylase, fremgangsmaade til dens fremstilling samt praeparat indeholdende beta-amylasen
DK0770/86 1986-02-19

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US (1) US4970158A (fr)
EP (1) EP0234858B1 (fr)
JP (1) JPS62201577A (fr)
AT (1) ATE97954T1 (fr)
CA (1) CA1304030C (fr)
DE (1) DE3788291T2 (fr)
DK (1) DK160563C (fr)
ES (1) ES2059362T3 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5312739A (en) * 1992-05-28 1994-05-17 National Science Council Production of high-maltose syrup and high-protein byproduct from materials that contain starch and protein by enzymatic process
US20040067570A1 (en) * 2001-02-06 2004-04-08 Pekka Kekki Process for the extraction of beta-amylase
US7638151B2 (en) 2003-03-10 2009-12-29 Danisco Us Inc. Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same
EP2422630A1 (fr) 2010-08-24 2012-02-29 Corn Products International, Inc. Production d'isomaltooligosaccharides et utilisations associées
US20140186495A1 (en) * 2011-08-02 2014-07-03 Yuan Yao Extraction, purification, and processing of phytoglycogen
WO2019081976A2 (fr) 2017-10-25 2019-05-02 Basf Se Enzymes bêta-amylase
WO2020219450A1 (fr) 2019-04-23 2020-10-29 Basf Se Variants de bêta-amylase

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE38507E1 (en) * 1989-09-27 2004-04-27 Novozymes A/S Antistaling process and agent
EP1350432A1 (fr) 2002-04-05 2003-10-08 Puratos N.V. Méthode pour retarder le rassissement des produits de boulangerie par addition d'une protéase thermostable
WO2012010592A1 (fr) 2010-07-21 2012-01-26 Novozymes A/S Procédé de préparation d'un produit cuit contenant une amylase anti-rassissement et une peptidase
EP2797430A1 (fr) 2011-12-26 2014-11-05 DuPont Nutrition Biosciences ApS Utilisation de l'enzyme amylase
CN114072519A (zh) * 2019-04-15 2022-02-18 公立大学法人大阪 新型β淀粉酶及其应用和制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3868464A (en) * 1970-10-27 1975-02-25 Meiji Seika Kaisha Production of maltose with amylases produced by streptomyces
US4560651A (en) * 1981-04-20 1985-12-24 Novo Industri A/S Debranching enzyme product, preparation and use thereof
US4604355A (en) * 1983-03-25 1986-08-05 Novo Industri A/S Maltogenic amylase enzyme, preparation and use thereof
US4647538A (en) * 1984-09-18 1987-03-03 Michigan Biotechnology Institute Thermostable beta-amylase

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3804718A (en) * 1971-04-01 1974-04-16 Hayashibara Co Method of producing beta-amylase by bacterial fermentation
JPS4867487A (fr) * 1971-12-17 1973-09-14

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3868464A (en) * 1970-10-27 1975-02-25 Meiji Seika Kaisha Production of maltose with amylases produced by streptomyces
US4560651A (en) * 1981-04-20 1985-12-24 Novo Industri A/S Debranching enzyme product, preparation and use thereof
US4604355A (en) * 1983-03-25 1986-08-05 Novo Industri A/S Maltogenic amylase enzyme, preparation and use thereof
US4647538A (en) * 1984-09-18 1987-03-03 Michigan Biotechnology Institute Thermostable beta-amylase

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5312739A (en) * 1992-05-28 1994-05-17 National Science Council Production of high-maltose syrup and high-protein byproduct from materials that contain starch and protein by enzymatic process
US20040067570A1 (en) * 2001-02-06 2004-04-08 Pekka Kekki Process for the extraction of beta-amylase
US7399622B2 (en) 2001-02-06 2008-07-15 Danisco Sugar Oy Process for the extraction of β-amylase
US7638151B2 (en) 2003-03-10 2009-12-29 Danisco Us Inc. Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same
US7993689B2 (en) 2003-03-10 2011-08-09 Danisco Us Inc. Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same
US8715755B2 (en) 2003-03-10 2014-05-06 Danisco Us Inc. Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same
EP2422630A1 (fr) 2010-08-24 2012-02-29 Corn Products International, Inc. Production d'isomaltooligosaccharides et utilisations associées
US20140186495A1 (en) * 2011-08-02 2014-07-03 Yuan Yao Extraction, purification, and processing of phytoglycogen
US10918124B2 (en) * 2011-08-02 2021-02-16 Purdue Research Foundation Extraction, purification, and processing of phytoglycogen
WO2019081976A2 (fr) 2017-10-25 2019-05-02 Basf Se Enzymes bêta-amylase
WO2020219450A1 (fr) 2019-04-23 2020-10-29 Basf Se Variants de bêta-amylase

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Publication number Publication date
ATE97954T1 (de) 1993-12-15
DK77086A (da) 1987-08-20
DK77086D0 (da) 1986-02-19
ES2059362T3 (es) 1994-11-16
DK160563C (da) 1991-09-02
EP0234858A2 (fr) 1987-09-02
JPS62201577A (ja) 1987-09-05
DE3788291T2 (de) 1994-03-31
DK160563B (da) 1991-03-25
DE3788291D1 (de) 1994-01-13
EP0234858B1 (fr) 1993-12-01
EP0234858A3 (en) 1988-08-03
CA1304030C (fr) 1992-06-23

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